Gram-negative septicemia, the clinical consequence of gram-negative bacterial invasion into the blood stream or tissue, occurs at a frequency of between 71,000 and 300,000 cases annually in the United States. Approximately forty percent of septicemia cases are associated with septic shock, a serious and rapidly developing complication of septicemia. Septic shock is characterized by hypotension, oliguria, coagulation defects, respiratory failure, and death.
The complex array of inflammatory responses in animals elicited by gram-negative bacteria is believed to be provoked by lipopolysaccharides (LPS) present in the outer membranes of these bacteria. Typically, an LPS molecule consists of an O polysaccharide, an R-core oligosaccharide, and lipid A. The structure of lipid A is highly conserved across a wide range of bacterial genera and is generally believed to be responsible for most of the biological activities of LPS. LPS is believed to provoke a number of both toxic and beneficial inflammatory responses and is believed to be responsible for the interaction of bacteria with target cells, which include macrophages, neutrophils and endothelial cells. While the toxic responses include hypotension, coagulation disturbances and death, beneficial responses include enhanced antibody synthesis, mobilization of phagocytes and acute phase protein synthesis.
Presently, there are no vaccines to immunize at-risk populations against septicemia. Current treatment of septicemia relies heavily on early diagnosis followed by antibiotic therapy. Septic shock patients are treated symptomatically with concurrent antibiotic therapy because of the rapid onset and severity of the symptoms associated with gram-negative septicemia. Current treatments consist of first administering a best guess antibiotic followed by identification through blood cultures and adjustment of antibiotic treatment; however, such a treatment regime does not inactivate the LPS which continue to induce their toxic effects.
Immunotherapy has been suggested as a treatment for gram-negative septicemia. Ziegler et al. (New Eng. J. Med. 307: 1225-1230, 1982) conducted a randomized, controlled trial using human anti-core LPS antiserum which demonstrated that immune serum reduced bacteremic mortality. The use of a human polyclonal antisera has significant drawbacks. In addition, the standardization of such preparations is difficult and there is the risk of transmitting viral infections such as HIV or hepatitis. Preparations of monoclonal antibodies have been used to treat septicemia, but the efficacy of such treatment is not undisputed.
Neutrophils have been shown to contain an enzyme, acyloxyacyl hydrolase (AOAH), that partially deacylates the lipid A portion of Salmonella typhimurium LPS by removing secondary fatty acyl chains (Hall and Munford, Proc. Natl. Acad. Sci. USA 80: 6671-6675, 1983). AOAH has been shown to contain two disulfide-linked subunits with apparent molecular weights of 50,000 and between 14,000-20,000 (Munford and Hall, J. Biol. Chem. 264: 15613-15619, 1989). The large subunit has been shown to be glycosylated. Munford and Hall (Science 234: 203-205, 1986) showed that when rabbits were injected intradermally with AOAH-treated LPS, and subsequently challenged with an intravenous injection of untreated LPS there was little or no hemorrhagic necrosis of the skin at the intradermal injection site. In contrast, rabbits that were initially injected with untreated LPS exhibited necrosis. AOAH-treated LPS, while reducing the LPS toxicity 100-fold, was found to reduce stimulation of mitogenesis of .beta.-lymphocytes by only a factor of 12. Munford and Hall (U.S. Pat. No. 4,929,604) have suggested that AOAH may be useful in treating/preventing septic shock caused by gram-negative bacteria.
The purification of AOAH from neutrophils as reported by Munford and Hall (J. Biol. Chem., ibid.) has a number of disadvantages. Although AOAH has been purified from a cultured human premyelocytic cell line, HL-60, and peripheral blood monocytes and neutrophils, it is only a trace protein, accounting for less than 0.001% of the protein in cell lysate. The purification method is a labor intensive, multi-step protocol that does not lend itself to commercial scale-up. A preparation of 9.5 .mu.g of AOAH, accounting for 7.8% of the original activity, was purified from approximately 5.times.10.sup.11 cells grown in 150 liters of media over a period of 2 months (Munford and Hall, ibid.). In addition, not all HL-60 cell lines are inducible to produce AOAH and AOAH specific activity fluctuates 2-3 fold as the cells are passaged. Purification of AOAH from peripheral blood neutrophils and monocytes has the risk of co-purifying infective agents such as the hepatitis viruses, HIV-1 and other viral agents, and the availability of large amounts of blood is not always assured.
There is therefore a need in the art for a method of producing relatively large amounts of pure preparations of AOAH, which would be useful as, inter alia, a therapeutic agent in the treatment of septic shock and for producing LPS vaccines. The present invention fulfills these and other related needs through the use of recombinant DNA technology, thus eliminating the problem of viral contamination and providing commercially feasible quantities of biologically active recombinant AOAH.